Method of ameliorating or preventing septic shock using a monoclonal antibody specific to cachectin/tumor necrosis factor

ABSTRACT

The present invention pertains to the novel hybridoma SDW18.1.1, hybridomas obtained from SDW18.1.1, monoclonal antibodies obtained from such hybridomas and derivatives of such monoclonal antibodies. The novel hybridomas are formed by fusion of cells from a mouse myeloma line and spleen cells from a mouse previously immunized with cachectin/TNF. Diagnostic and therapeutic utilities for the monoclonal antibodies and their derivatives are proposed, and testing procedures, materials in kit form and pharmaceutical compositions are likewise set forth.

RELATED APPLICATIONS

The present application is a continuation of application Ser. No.07/286,477, filed Dec. 19, 1988, now abandoned, which is acontinuation-in-part of application Ser. No. 766,852, filed Aug. 16,1985 now abandoned, which is a continuation-in-part of Ser. No. 414,098,filed Sep. 7, 1982, now U.S. Pat. No. 4,603,106, issued Jul. 29, 1986,which is in turn a continuation-in-part of Ser. No. 351,290, filed Feb.22, 1982, now abandoned, which is in turn a continuation-in-part of Ser.No. 299,932, filed Sep. 8, 1981, also abandoned, in all of which atleast one of the Applicants herein is a co-inventor. Applicants claimthe benefit of these applications under 35 U.S.C. Section 120.

RELATED PUBLICATIONS

The Applicants are authors or co-authors of several articles directed tothe subject matter of the present invention. These articles are insupplementation to those articles listed in U.S. Pat. No. 4,603,106,which earlier articles are incorporated herein by reference. (1)Applicant Cerami co-authored with B. Beutler, J. Mahoney, N. Le Trangand P. Pekala! "Purification of Cachectin, a LipoproteinLipase-Suppressing Hormone Secreted By Endotoxin-Induced RAW 264 7Cells", J. EXP. MED. 161 at 984-995 (May, 1985); (2) Applicant Ceramico-authored with J. R. Mahoney, B. Beutler, N. Le Trang, W. Vine, and Y.Ikeda! "Lipopolysaccharide-Treated RAW 264.7 Cells produce a MediatorWhich Inhibits Lipoprotein Lipase in 3T3-L1 Cells", J. IMMUNOL. 134 (3)at 1673-1675 (March, 1985); (3) Applicant Cerami co-authored with P. J.Hotez, N. Le Trang, and A. H. Fairlamb! "Lipoprotein Lipase Suppressionin 3T3-L1 Cells by a Haematoprotozoan-Induced Mediator From PeritonealExudate Cells", PARASITE IMMUNOL. (Oxf.) 6:203 (1984); (4) ApplicantCerami co-authored with B. Beutler, D. Greenwald, J. D. Hulmes, M. ChangY.-C. E. Pan, J. Mathison and R. Ulevitch! "Identity of Tumor NecrosisFactor and Macrophage-Secreted Factor Cachectin", NATURE 316:552-554,(1985); (5) Applicant Cerami co-authored with B. Beutler, F. M. Torti,B. Dieckmann and G. M. Ringold! "A Macrophage Factor Inhibits AdipocyteGene Expression: An In Vitro Model of Cachexia", SCIENCE 229:867-869,(1985); (6) Applicant Cerami co-authored with B. Beutler and I. W.Milsark! "Passive Immunization Against Cachectin/Tumor Necrosis Factor(TNF) Protects Mice From the Lethal Effect of Endotoxin", SCIENCE229:869-871, (1985); (7) Applicants Cerami and Wolpe co-authored with K.J. Tracey, B. Beutler, S. F. Lowry, J. Merryweather, I. W. Milsark, R.J. Hariri, T. J. Fahey III, A. Zentella, J. D. Albert and G. T. Shires!"Shock And Tissue Injury Induced By Recombinant Human Cachectin",SCIENCE 234:470-474 (1986); and (8) Applicant Cerami co-authored with K.J. Tracey, Y. Fong, D. G. Hesse, K. R. Manogue, A. T. Lee, G. C. Kuo andS. F. Lowry! "Anti-Cachectin/TNF Monoclonal Antibodies prevent SepticShock During Lethal Bacteraemia", NATURE 330:662-664 (Dec. 17, 1987).All of the above listed articles are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention is directed to new hybrid cell lines and morespecifically to hybrid cell lines for production of monoclonal antibodyto cachectin/TNF, to the antibody so produced, to antibodies raisedthereto, and to diagnostic and therapeutic methods and compositionsemploying these antibodies.

The development of monoclonal antibodies has made possible newdiagnostic and therapeutic techniques. Monoclonal antibodies arehomogeneous immunoglobulins of well-defined chemical structure incontrast to polyclonal antibodies which are heterogeneous mixtures ofimmunoglobulins. A characteristic feature of monoclonal antibodies ishigh specificity and reproducibility of function.

The technique for producing monoclonal antibodies from hybridized cells(hybridomas) was first described by Kohler and Milstein NATURE256:495-497 (1975)!. The hybridomas are produced by fusion of mousemyeloma cells with spleen cells from immunized mice or rats. Clones ofthe hybridoma cells (cells arising from a single parent plasmacytomaspleen fusion cell) are then tested for their ability to produce thedesired monoclonal antibody. Each clone produces only a single antibodytype directed against a single antigen only and is therefore highlyspecific. In contrast to conventional (polyclonal) antibody preparationswhich typically include different types of antibodies directed againstdifferent sets of determinants (sites) on the same antigen, monoclonalantibody preparations are directed only against a single determinant.

Although the general procedures for preparing hybridomas are known,there is no certainty that the desired hybridoma will be obtained, thatthe hybridoma obtained will produce the desired antibody and that theantibody obtained will have the desired degree of specificity. The levelof success is chiefly dependent upon the type of antigen employed duringthe immunization procedure and the selection technique employed forisolating the desired hybridoma.

One of the areas of interest for using monoclonal antibodies is in thediagnosis and treatment of biochemical derangements that occur inmammalian hosts responding to invasive stimuli. These gross imbalancesof host physiology are generally manifested as a wasting of the body(cachexia) which itself may threaten the integrity of the host. Thesemetabolic disorders seem to be mediated largely by the immune system.For example, in response to invasive stimuli, the reticuloendothelialcells and lymphocytes secrete cytokines which are capable of alteringhost metabolism. These cytokines include for example, Interleukin-1,Interleukin-2, lymphotoxin, gamma-interferon and the substance known aseither cachectin or tumor necrosis factor (TNF).

Systemic deficiency of the anabolic enzyme lipoprotein lipase (LPL)activity has been observed in cachectic animals. Deficiency of LPLactivity has also been observed in mice after administration ofendotoxin (lipopolysaccharide, LpS). In contrast, deficiency of LPLactivity has not been observed in mice genetically resistant to LPS.Resistance to endotoxin-induced LPL deficiency could be overcome byadministration of serum obtained from endotoxin-sensitive animals whichhad been previously injected with LPL. The active factor in this serumwas termed "cachectin" because of its involvement in the pathogenesis ofcachexia M. Kawakami et al., PROC. NATL. ACAD. SCI., (USA), 79:912-916(1982)!.

The existence of a factor which caused hemorrhagic necrosis of tumors,in the serum of endotoxin-treated animals previously infected withMycobacterium bovis strain Bacillus Calmette-Guerin was also observed.The active principal in this serum was termed "tumor necrosis factor" E.A. Carswell et al., PROC. NATL. SCI. (USA), 72:3666-3670 (1975)!.

The potent tumor necrosis factor activity of cachectin in vitro and DNAsequencing of the primary structure of cachectin and tumor necrosisfactor confirmed that these polypetides are homologous molecules andthat their bioactivities are both derived from a highly conservedprotein. Accordingly, the term "cachectin/TNF" will be used whenreferring to these factors herein.

Cachectin/TNF is a polypeptide hormone composed of subunits having arelative molecular mass of 17,000 arranged in dimeric, trimeric orpentameric form depending upon the species and the method of isolation.When administered to animals in moderate amounts, cachectin/TNF inducesa state of anorexia and ensuing weight loss. Cachectin/TNF also seems toplay a major role in the pathogenesis of Gram-negative(endotoxin-induced) shock. Not only does the administration of largedoses of cachectin/TNF directly mimic the clinical syndrome produced byendotoxemia K. J. Tracey et al., SCIENCE 234:470-474 (1986)!, butpassive immunization against cachectin/TNF substantially mitigates thelethal effect of endotoxin K. J. Tracey et al., NATURE330:662-664(1987)!. Cachectin/TNF has the ability to substantiallysuppress the activity of the anabolic enzyme LPL and is capable ofpreventing the differentiation of fat cells and increasing the uptake ofglucose in muscle cells. Cachectin/TNF demonstrably lacks leukocyteactivator activity which characteristic distinguishes it fromInterleukin-1. Similarly, the ability of cachectin/TNF to significantlysuppress LPL activity distinguishes it from Interleukin-2. Thesefindings were set forth in copending parent application Ser. No.766,852, the disclosure of which is incorporated herein by reference.

Most cell types express specific high-affinity cell-surface receptorsfor cachectin/TNF, but the consequences of binding the cytokine arediverse and often cell- and species-specific. Certain cell types showmarked sensitivity to the cytotoxic effects of cachectin/TNF whileothers bind the cytokine but are not deleteriously affected. Other celltypes respond to cachectin/TNF treatment with specific and wellcoordinated changes in the activity, expression, synthesis, or releaseof surface or cytosolic proteins, enzymes, or further physiologicalmediators. Much research over the past few years has focused ondissecting apart these differential biological effects of cachectin/TNFand an important step in this direction would be the identification andcharacterization of cachectin/TNF receptors from various cellularsources.

Accordingly, there exists a need for antibodies in relatively pure formfor the detection and study of cachectin/TNF. More particularly, thereexists a need for monoclonal antibodies to cachectin/TNF for use in thediagnosis and treatment of cachexia and related diseases.

SUMMARY OF THE INVENTION

The present invention pertains to the novel hybridoma SDW18.1.1,hybridomas obtained from SDW18.1.1, monoclonal antibodies obtained fromsuch hybridomas, derivatives of such monoclonal antibodies and the useof such monoclonal antibodies and their derivatives of diagnostic andtherapeutic methods and compositions. The novel hybridomas are formed byfusion of cells from a mouse myeloma line and spleen cells from a mousepreviously immunized with cachectin/TNF.

Accordingly, it is a principal object of the present invention toprepare a monoclonal antibody to cachectin/TNF.

It is a further object of the present invention to employ the monoclonalantibody to detect and study as aforesaid the activity of cachectin/TNF.

It is a still further object of the present invention to employ themonoclonal antibody as aforesaid to diagnose and treat biologicalderangements in which cachectin/TNF is implicated.

It is a still further object of the present invention to prepareadditional antibodies from the monoclonal antibody as aforesaid whichmay be used in further diagnostic and therapeutic settings because oftheir ability to bind to the cellular receptor for cachectin/TNF.

Other objects and advantges will become apparent to those skilled in theart from a consideration of the ensuing description.

DETAILED DESCRIPTION

The present invention concerns the preparation and use of a monoclonalantibody to cachectin/TNF. The particular monoclonal antibody preparedand discussed herein was raised against human tumor necrosis factor. Itis understood however that the preparation and application of theantibody described herein extends to monoclonal antibodies based onother sources of cachectin/TNF and is therefore, intended to encompasssuch variations in source within its scope.

The novel monoclonal antibodies of the present invention provide a hightiter, reproducible, biological reagent for the assay of cachectin/TNF.Fluids or tissues from a variety of mammals can be screened byradioimmunoassay, enzyme immunoassay, immunofluorescence, complementfixation, immunoprecipitation or any reaction which depends uponantibody recognition of antigen for the detection of cachectin/TNF.Furthermore, the novel monoclonal antibodies of the present inventionare useful in the diagnosis and treatment of cachexia and relateddiseases.

The hybridoma SDW18.1.1 was deposited at the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 for patentpurposes as defined in M.P.E.P. 608.01(p) on Oct.15, 1986 and was giventhe A.T.C.C. Accession No. HB 9228.

In general, the method of preparing the hybridoma of the presentinvention comprises the following steps: (a) immunizing mice withcachectin/TNF; (b) removing the spleens from the mice and preparing animmunized spleen-cell suspension; (c) fusing the suspended spleen cellswith mouse myeloma cells from a suitable cell line using a fusionpromoter; (d) diluting and culturing in separate wells the mixture ofunfused spleen cells, unfused myeloma cells, and fused hybrid cells in aselective medium which will not support the unfused myeloma cells for atime sufficient to allow the death of the unfused cells; (e) evaluatingthe supernatant in each well containing the hybridoma for the presenceof antibodies to cachectin/TNF; and (f) selecting and cloning hybridomasproducing the desired monoclonal antibodies.

The immunization schedule and the concentration of cachectin/TNF shouldbe such that useful quantities of primed splenocytes are obtained.Freund's adjuvant may be used to prime the immune system. Aftercentrifugation and washing, the immunized spleen cells are ready to befused with mouse myeloma cells. Suitable fusion promoters arepolyethylene glycol (PEG) and dimethyl sulfoxide (DMSO).

The myeloma cell lines chosen should preferably be the drug resistanttype such as the 8-azaguanine resistant cell lines which lines aredeficient in the enzyme hypoxanthine guanine ribosyl transferase (HPRT)or thymidine kinase. The lack of these specific enzymes in these linesmakes it impossible for these myeloma cells to incorporate exogenouslysupplied hypoxanthine or thymidine. Endogenous DNA synthesis may beblocked by use of aminopterin. Thus, unfused myeloma cells will not besupported by hypoxanthine, aminopterin, and thymidine (HAT) medium.Similarly, the unfused nonmalignant immunized spleen cells have only afinite number of generations and will not survive in HAT for more than afew days. The fused hybrid cells, on the other hand, will continue toreproduce because they possess the malignant quality of the myelomaparent and are able to survive in the selective medium by virtue ofmetabolic pathways deriving from the spleen cell parent.

The hybridomas may be cloned using the limited dilution method or thesolid gel media method. The preferred cloning method is the limiteddilution method. In this method, a hybridoma suspension isproportionately divided among a series of sterile wells. Visualappearance of colonies usually takes one to two weeks. The wells havingthe fewest hybridomas, showing single clones, are then evaluated forantibody production.

Once the hybridoma of choice has been selected and cloned, the desiredmonoclonal antibody may be produced in vitro or in vivo. The purestmonoclonal antibody is produced in vitro by culturing the desiredhybridoma in a suitable medium for a suitable length of time followed byrecovering the desired antibody from the supernatant. The suitablemedium and the suitable length of culturing time are known or arereadily determined. This in vitro technique produces monoclonal antibodyessentially free from other nonspecific antihuman immune globulins.However, this method may not produce a sufficient quantity of asufficient concentration of monoclonal antibody for some purposes sincethe concentration of antibody obtained is relatively low.

Much higher concentrations (high titer) of slightly less pure monoclonalantibody may be produced using the in vivo method. In this method, thedesired hybridoma is injected into mice, preferably syngenic orsemisyngenic mice, causing formation of antibody-producing tumors aftera suitable incubation time. These tumors will produce a relatively highconcentration of the desired antibody in the bloodstream and peritonealexudate (ascites) of the host mouse. Although these host mice also havenormal antibodies in their blood and ascites, the concentration of thesenormal antibodies is low and these normal antibodies are not usuallyantihuman in their specificity.

The hybridomas of the present invention may also be used as a source ofgenetic material. For example, the hybridomas may be fused with othercells to provide still other novel hybridomas having the same secretorycapabilities as SDW18.1.1 and providing antibodies having the samespecificity. Such fusion of the subject hybridoma to other cells mayresult in the production of antibodies having different heavypolypeptide chains, providing other classes or subclasses of antibodiessuch as IgM, IgA, IgG₂, IgD, IgE, etc.

Although only a single novel hybridoma producing a single monoclonalantibody against cachectin/TNF antigen is described, Applicants intendthe present invention to encompass all monoclonal antibodies whichexhibit the characteristics described herein. The monoclonal antibody ofthe present invention to cachectin/TNF belongs to the subclass IgG1. Theother classes and subclasses of IgG antibodies differ from one anotherin their "fixed" regions, i.e. areas having the same amino acidsequences. However, these antibodies will also have a "variable" regionwhich is functionally identical, i.e. antigen specific, regardless ofwhich antibody class or subclass to which the antibody belongs. Hence, amonoclonal antibody exhibiting the characteristics described herein maybe of class IgM, IgA and so forth. Differences among these classes orsubclasses will not affect the selectivity of the reaction pattern ofthe monoclonal antibody but may affect the reaction of the antibody withother materials. Although the antibody of the present invention belongsto class IgG1, Applicants intend that all antibodies having the patternsof reactivity illustrated herein are included within the presentinvention regardless of the immunoglobulin class or subclass to whichthey belong. Furthermore, for many applications, the entire monoclonalantibody molecule need not be used but only a fragment of the moleculehaving intact antigen-binding sites will suffice.

Although only a single hybridoma is described here, Applicants intendthe present invention to encompass all methods for preparing themonoclonal antibodies described above employing the hybridoma techniquedescribed herein. One skilled in the art could follow the immunization,fusion, selection and cloning methods provided herein and obtain otherhybridomas capable of producing monoclonal antibodies having thereactivity characteristics disclosed herein. Since the novel hybridomaproduced from a known mouse myeloma cell line and spleen cells from aknown species of mouse is best characterized by description of theantibody produced by the hybridoma, all hybridomas producing antibodyhaving the reactivity characteristics described above are includedwithin the subject invention, as are methods for making this antibodywhich employ the hybridoma.

It has now been found that cachectin is the principal mediator in bothendotoxin-induced shock as well as tumor necrosis. Nature, Vol. 320, No.6063, pp. 584-588, 17 Apr. 1986!Applicants have successfully shown thisto be true through passive immunization of baboons against endogenouscachectin. The baboons were subsequently infused with an LD₁₀₀ dose oflive Escherichia coli. Control animals (not immunized against cachectin)developed hypotension followed by lethal renal and pulmonary failure.Neutralizing monoclonal anti-cachectin antibody fragments F(ab')₂ !administered to baboons only one hour before bacterial challenge,protected against shock but did not prevent critical organ failure.Complete protection against shock, vital organ dysfunction, persistentstress hormone release and death was conferred by administration ofcachectin monoclonal antibodies 2 hours before bacterial infusion. Theseresults indicate that cachectin is a mediator of fatal bacteremic shock,and suggest that cachectin antibodies offer a potential therapy forlife-threatening infection. Thus, through passive immunization withcachectin antibodies, the deleterious effects of caohectin, such asseptic shock, can be prevented or at least reduced. This invention thuscontemplates a composition containing cachectin antibody for passiveimmunization of mammals against the effects of endotoxin. Additionally,this invention further contemplates an assay kit for the detection ofhuman tumor necrosis factor, said kit comprising monoclonal antibody orantibody fragments reactant to cachectin, as well as other reagents anddirections for use of such kit.

Accordingly, the present invention is also directed to in vivo and invitro methods of diagnosis as well as therapy employing the monoclonalantibody to cachectin/TNF as well as anti-idiotype antibodies raisedthereto. These techniques may be employed using the cachectin/TNFantibody or the anti-idiotype antibody alone, or in combination withother antibodies. For many applications, the antibodies will be labeledwith a compound which imparts a detecting signal, providingcytotoxicity, providing for localizing electromagnetic radiation, or thelike. Labels may include radionuclides, enzymes, fluorescent moieties,toxins or the cytoxic fragment of toxins, particles, metals, metalloids,etc. The antibodies may be incorporated in liposome membranes ormodified with lipids so as to be incorporated in such membranes. Theantibodies by themselves or labeled may be used in in vitro diagnosisfor measuring the presence of antigens associated with cachectin/TNF,for in vivo diagnosis for introduction into a host, e.g., intravenously,in a physiologically acceptable carrier, e.g., phosphate bufferedsaline, or may be introduced for therapeutic purposes in the samemanner. Moreover, the antibodies of the present invention may beemployed in methods and compositions for assaying cachectin/TNF, fordiagnosing and treating disease states such as cachexia, septic shock,and related diseases and for the preparation of passive vaccines againstthese diseases. The amount of antibody employed will vary depending uponthe particular application. The use of antibodies for diagnostic andtherapeutic purposes has been extensively described in the literature.

Treatment of disease states such as cachexia may be accomplished byadministration of a therapeutically effective amount of cachectin/TNFantibody to an individual in need of such treatment. By selectivereaction with cachectin/TNF antigen, the effective amount ofcachectin/TNF antibody will neutralize the excess of antigen, thusameliorating the effects of the excess, such as undesirable weight loss.Diagnostic and therapeutic compositions comprising effective amounts ofcachectin/TNF antibody in admixture with diagnostic or pharmaceuticallyacceptable carriers, respectively, are also included within the presentinvention.

In addition to the discovery of the above-described hydridoma cell lineand the monoclonal antibodies produced therefrom, it has also beendiscovered that another set of polyclonal, polyspecific antibodies canbe produced which are reactive with the monoclonal antibodies describedherein. These polyclonal antibodies are, in effect, antibodies againstantibodies and a subset is called anti-idiotype antibodies. Byintroducing anti-idiotype antibodies into the cachectin/TNF-TNF receptorreaction, a competitive reaction is set up between the anti-idiotype andthe cachectin/TNF molecule which may result in less binding of thecachectin/TNF molecule with its cellular receptor. Thus, theanti-idiotype antibody can block the cachectin/TNF ligand complex frombinding with its receptor, thereby possibly altering the deleteriouseffects of cachectin/TNF.

The present invention is further illustrated by the following exampleswhich are not intended to limit the effective scope of the claims.

EXAMPLE

Recombinant human tumor necrosis factor (hTNF) (Genentech, Inc.) wasemulsified in complete Freund's adjuvant. The equivalent of 17micrograms of hTNF was injected subcutaneously into two mice(C57B1/6×Balb/c F1 female, Charles River Kingston). One month later, thespleens of these two mice were surgically exposed and 10 micrograms ofhTNF in 100 microliters of phosphate-buffered saline ("PBS") wereinjected into each mouse intrasplenically. The spleens were placed backinto the body cavities and the incisions were closed.

Three days later, the spleens were removed and carefully teased apart inDulbecco's modified Eagle's medium (Grand Island Biological Company,Grand Island, N.Y.) with 4.5 grams per liter glucose and.10% fetalbovine serum to yield a single-cell suspension.

These immunized spleen cells were fused with the P3-×63-Ag8.653 myelomacell line (American Type Culture Collection) utilizing the protocoldescribed in Monoclonal Antibodies, R. Kennett, K. Bechtol and T.McKearn (Eds.), plenum press (1980). The fused cells were plated ontomacrophage feeder layers using the protocol described by Fazekas de St.Groth and Scheidegger, J. Immunol. Methods, 35,1-21 (1980), and allowedto grow until macroscopic colonies were observed.

Colonies were tested for the production of antibody against hTNF usingan immunoblotting assay. Nitrocellulose sheets were immersed in PBS andblotted dry. A quantity of 5 microliters of hTNF (initially obtainedfrom Genentech, Inc., and later obtained from Chiron Corp.) at aconcentration of 0.1 microgram per milliliter were dotted onto spots onthe nitrocellulose plate through wells in a Bio-Rad dot-blottingapparatus. The nitrocellulose was blocked using a solution of 1% bovineserum albumin in PBS. A quantity of 100 microliters of the supernatantabove the cultured hybridoma colonies was added to each dot-blot andwell, and incubated for 45 minutes. The clones which reacted positivelywere visualized by the subsequent use of a VECTASTAIN (trademark)avidin-biotin-peroxidase system (Vector, Inc.) according to theinstructions of the manufacturer.

Initially, thirteen positive clones were identified. Four clonesappeared to show false positives because they also showed positive inthe absence of antigen. One clone (#23) showed positive with antigenmaterial obtained from Genentech, Inc. but not with antigen materialobtained from Chiron Corp. Four clones (#27, #37, #42 and #156) appearedto lose activity during further propagation. These four clones and threeother clones (#299, #433 and #471) were set aside for furthercharacterization.

One clone (#18) exhibited consistent reactivity and retained thisreactivity during subcloning. Subcloning was achieved by limiteddilution on macrophage feeder layers. Dilute suspensions of a givenclone were plated onto 96-well plates such that less than a third of thewells were positive for growth.

Each subcloning was designated by a period. Hence, the designation--18.1.1--means that clone #18 was subcloned twice and the first wellwhich showed growth was chosen for further subcloning each time. Thesubject hybrid antibody was demonstrated by standard techniques to be ofclass IgG1.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A method of ameliorating or preventing septic shock which comprises administering to a mammalian patient suspected of having a need for such administration an effective amount of a monoclonal antibody exhibiting the characteristics of a monoclonal antibody as produced by hybridoma cell line having the identifying characteristics of A.T.C.C. Accession No. HB
 9228. 2. The method of mitigating claim 1 wherein the monoclonal antibody is produced from a hybridoma cell line having the identifying characteristics of A.T.C.C. Accession No. HB
 9228. 